The single plane illumination microscopy (SPIM) technique, in which illumination of the sample occurs in layers, allows faster capture of image data with less sample impact than, for example, with spot scanning of a sample. One known area of application of SPIM technology is the field of fluorescence microscopy, in which fluorophores in the sample are excited with laser light. In SPIM technology, excitation takes place here only in a plane through which a sheet of illuminating light (also called a “light band”) is passed. Damage to the sample due to illuminating light in other planes is thereby avoided.
A microscope operating on the SPIM principle (selective plane illumination microscope) is described, for example, in Lindek et al., Journal of Modern Optics, 1999, Vol. 46, No. 5, 843-858.
An optical apparatus operating in accordance with the SPIM method is described in DE 102 57 423 A1. With this microscope, a sample is illuminated with a thin light band while observation occurs perpendicularly to the plane of the illuminating light band. Here illumination and detection occur via two separate optical beam paths each having a separate optical system, in particular having two separate, mutually perpendicular objectives. The light band is generated by an illumination objective and by a cylindrical optic placed in front of it. For image acquisition, the sample is moved through the light band (which is stationary relative to the detector) in order to acquire fluorescent light and/or scattered light in layers using a planar detector. The layer image data thereby obtained can then be assembled into a data set corresponding to a three-dimensional image of the sample. This document does not disclose manipulation of a sample.
DE 10 2004 034 957 A1 discloses an arrangement for microscopic observation of a sample via a microscope objective in whose housing, outside the lens optic, light guides for the light illuminating the sample are provided. The illuminating light firstly proceeds parallel to the optical axis of the objective inside the light guide and then strikes small-aperture reflectors, attached to the objective housing, which focus the illuminating light with the aid of additional imaging elements, perpendicularly to the optical axis of the microscope objective and thus perpendicularly to the observation direction, into the sample. Here as well, the sample is illuminated in planar fashion using the SPIM principle. The utilization of a microscope objective configured in this fashion does allow elimination of the use of a further objective for the illuminating light. The specific configuration of this special objective, however, with additional light guides and reflectors, is technically very complex and costly.
DE 20 2011 110 077 U1 discloses an arrangement for illuminating a sample in the context of SPIM microscopy. The arrangement comprises a light source for generating a light bundle, means for generating a light band from the light bundle, and at least one objective which comprises an optic that is configured and intended to deliver detected light, proceeding from the sample, directly or indirectly to a detector. The arrangement furthermore comprises a deflection device, downstream from the optic of the objective, for deflecting the light band.
Deflection devices of this kind have so far been laboriously manufactured in one piece, and are usually mounted in very cumbersome fashion. In particular, the deflection devices so far manufactured are usually manufactured using very complex production methods that are not suitable for series production.